Cells of the innate immune system, especially dendritic cells (DCs), direct the differentiation of naïve CD4+ T cells into functionally distinct Th1, Th2, ThIL-17 (also known as Th17) or regulatory T (Tregs) cell subtypes. Activation of immature DCs through binding of conserved microbial molecules to pathogen recognition receptors (PRRs), such as Toll-like receptors (TLRs) and integrins, is accompanied by DC maturation and homing to the lymph nodes, where the mature DCs present antigen (Ag) to naïve T cells. Activation of dendritic cells by pathogen derived molecules plays a critical role in regulating the differentiation of naïve CD4+ T cells into distinct T cell subtypes. Th1 cells confer protection against intracellular infection but are also associated with inflammatory responses and autoimmune disease, whereas Th2 cells are involved in allergic responses. IL-17 producing T cells (ThIL-17 or Th17) are a pathogenic subset of T cells which are characterised by the production of IL-6, TNF-alpha and, in particular, IL-17 and IL-17F, important pro-inflammatory cytokines which contribute to autoimmune disease. Treg cells are capable of suppressing Th1, Th2 and Th17 responses.
Immune Mediated Conditions
Multiple sclerosis (MS) is an autoimmune disease that affects the central nervous system. Individuals with this disease have autoreactive T cells (T cells that recognize self antigens), which together with interleukin (IL)-1beta and tumour necrosis factor (TNF) alpha, participate in the formation of inflammatory lesions along the myelin sheath of nerve fibres. The cerebrospinal fluid (CSF) of patients with MS contains activated T cells, which infiltrate the brain tissue and cause the characteristic inflammatory lesions, destroying the myelin. Experimental autoimmune encephalomyelitis (EAE) is an animal model for MS. It is induced in mice or rats by injection of mylein basic protein (MBP) or myelin oligodendrocyte glycoprotein (MOG) or peptides thereof with complete Freund's adjuvant. The disease can also be induced by transfer of MBP or MOG-specific T cells that secrete IL-17 (called ThIL-17 cells or Th17 cells). The animals develop cellular infiltration of the myelin sheaths of the central nervous system, resulting in demyelination and eventually paralysis. The clinical signs and pathological changes resemble MS.
Crohn's disease and ulcerative colitis are inflammatory bowel diseases in humans. These autoimmune diseases are inflammatory conditions of the intestine mediated by CD4+ T cells.
Many autoimmune and chronic inflammatory diseases have no satisfactory treatment and in most cases steroids and non-steroidal anti-inflammatory drugs are employed. However, these are non-specific and have side effects. More recently, drugs that inhibit key inflammatory cytokines, in particular tumour necrosis factor-alpha (TNF-alpha), have been developed. These include antibodies or soluble TNF receptors that are effective against certain autoimmune diseases, but are associated with side effects (including recurrent tuberculosis and cancers) and are limited to diseases where TNF-alpha is the key mediator of pathology. Another therapeutic approach is the direct administration of anti-inflammatory cytokines (e.g. IL-10), but this is compromised by the short half-life of cytokines in vivo. Alternative strategies employ agents that induce anti-inflammatory cytokines, such as IL-10, which will have a direct immunosuppressive effect in vivo. Molecules that promote the induction of suppressor or regulatory T cells have the potential to limit inflammatory Th1-mediated immune responses and also the T cells which secrete IL-17 (ThIL-17) which also mediate inflammatory responses in T cell mediated autoimmune responses.
Helminth Infection
Infection with Helminth parasites has been associated with immunosuppression and compromised T-cell responses. Infection with the liver fluke Fasciola hepatica induces highly polarized Th2 responses in mice, with high levels of the cytokines IL-4, IL-5 and IL-10 and also IgG1 antibodies. Infection with F. hepatica suppressed IFN-gamma secreting T cell (Th1 cell) responses and protection against the bacterial pathogen, Bordetella pertussis. Infection with F. hepatica also inhibited Th1 responses and protection against B. pertussis induced with a whole cell pertussis vaccine.
F. hepatica excretory/secretory (ES) components have also been shown to suppress Th1 responses induced by a whole cell B. pertussis vaccine. This suppression was shown to be reversed by an inhibitor of cathepsin L proteinase, this suggesting that cathepsin L proteinase was responsible for mediating the suppressive effect.
Further, cathepsin L proteinase purified from F. hepatica ES component suppressed Th1 response induced with Pw vaccine. These studies also demonstrated that the suppression induced by F. hepatica or the cathepsin L proteinase were mediated by IL-4 as suppression of the immune response was seen to be reversed in IL-4 defective mice.
The conclusion of the above studies was therefore that cathepsin L proteinase was responsible for, and mediated the suppressive effect of, F. hepatica and F. hepatica ES component and that the suppression was mediated through IL-4 induction.
A method of preventing the onset and progression of autoimmune diseases, inflammatory conditions or immune mediated disorders through the modulation of the T cell responses which are causative of these conditions would be highly advantageous in the prevention and treatment of these conditions.
The inventors of the present invention have surprisingly identified that the excretory/secretory (ES) component of Fasciola hepatica comprises compounds other than cathepsin L proteinase which mediate suppression of the immune response. While it was considered that suppression of the immune response by cathepsin L proteinase was mediated by IL-4 cytokine production, the present inventors have identified that the ES component mediates immune suppression through a number of alternative mechanisms which serve to modulate the immune response. In particular, the inventors have identified that the excretory/secretory (ES) component of Fasciola hepatica interacts with the cells of the innate immune system to modulate the immune response through the modulation of cytokine expression, in particular the upregulation of IL-10 cytokine levels, through the activation of dendritic cells into a phenotype which promotes a T regulatory phenotype (which produces IL-10) and which skews the immune response away from the production of T cells having a Th1 and/or ThIL-17 phenotype.
In particular, the inventors have identified that the ES component of F. hepatica stimulates the production of anti-inflammatory cytokines, such as IL-10, which can serve as potent modulators of the immune response.
Further, the inventors have also surprisingly shown that the ES fraction from F. hepatica can activate dendritic cells into a phenotype that promotes the production of regulatory T cells (Tregs), these Tregs in turn modulating the immune response, through the suppression of Th1 and ThIL-17 type responses. In particular, such dendritic cells have been shown to exhibit low expression levels of cell surface markers CD80, CD86, CD40 and MHC class II, while higher expression levels of CCR5 are observed over the expression levels of these cell surface markers on naïve dendritic cells. The IL-17 producing T cell subset secretes a cytokine profile including IL-17, IL-17F, IL-17H, IL-17F, IL-17A, IL-6 and TNF. IL-17 producing T (Th17 (ThIL-17)) cells, driven by IL-1 and IL-23 or IL-6 and TGF-beta, are a distinct subtype of inflammatory T cells from Th1 cells and are pathogenic in many autoimmune diseases and chronic inflammatory conditions.
These effects are independent to the immune modulatory effects which have been hitherto observed in relation to cathepsin L proteinase. Importantly, the immunomodulatory effects observed by the present inventors are not suppressed by cathepsin L proteinase inhibitors and are not IL-4 dependent. Without wishing to be bound by theory, the inventors therefore predict that the immunomodulatory effects mediated by the ES component are mediated, at least in part, by components or products derived from the ES component other than a cathepsin L proteinase.
Modulation of the response and cytokine profile expressed by a specific cell type of the immune system can lead, in turn, to a wider modulation of the overall immune response. The inventors have further surprisingly shown that the ES fraction from F. hepatica inhibits the induction of IFN-gamma, IL-17 producing Th1 cells (Th17 (ThIL-17) cells) and Th1 cells, through either the inhibition of IL-12 and IL-23, which promotes expansion of the Th1 and ThIL-17 cells, or by inhibiting the activation of Th1 and ThIL-17 cells, or by suppressing the function of Th1 and ThIL-17 cells.